Specific Activity Of Fraction Research Articles

Low molecular mass peptides generated by hydrolysis of casein impair rennet coagulation of milk

Previously it was shown that a low molecular mass fraction isolated from the proteose–peptone preparation of milk, fraction E, inhibited milk coagulation. Here, the composition and molecular mass of fraction E, and its effect on milk clotting parameters, was investigated to better understand its mechanism of action. Fraction E comprised casein-derived peptides of 1–3 kDa rich in phosphorus residues. Fraction E content increased substantially in milk from glands infected with Escherichia coli and Streptococcus dysgalactiae, and during storage of the milk. However, the specific activity of fraction E on milk clotting parameters was the same whether sampled from healthy, infected or stored milk. The inhibitory effect was reversible on adding 0.75 mm CaCl2, suggesting that chelation of Ca by fraction E was involved in the inhibitory mechanism. However, only partial recovery was achieved and an excess of Ca was required, suggesting the involvement of additional pathways in the process.

The potential for platelet-activating factor synthesis in brain: Properties of cholinephosphotransferase and 1-alkyl- sn-glycero-3-phosphate acetyltransferase in microsomal fractions of immature rabbit cerebral cortex

The synthesis of platelet-activating factor (PAF) was studied in microsomal fractions of cerebral cortices of 15-day-old rabbits. These included: a total microsomal fraction P 3, rough and smooth microsomes, R and S, and microsomal fraction P derived from isolated nerve cell bodies. Cholinephosphotransferase (CPT) generating PAF from alkylacetylglycerol had the highest specific activities in fractions R and P (24 and 6 times the homogenate values, based on membrane phospholipid content). This CPT activity differed from that which synthesized phosphatidylcholine as the latter was sensitive to dithiothreitol inhibition and was more readily inhibited by Triton X-100. As the CPT activity for PAF synthesis relies on the production of alkylacetylglycerol we studied the acetyltransferase which forms 1-alkyl-2-acetyl- sn-glycero-3-phosphate (AAGP). This enzyme had the highest specific activity in fraction R, followed by fractions P 3 and P. There was evidence that the acetyltransferase was more active in a phosphorylated form. NaF maximized the recovery of AAGP products in the assays. The pH optimium for acetylation was in a range of 8.0–9.0. Lyso PAF did not inhibit the formation of AAGP and the rates of formation of PAF by acetylation were less than 5% of values for AAGP synthesis. During AAGP formation there was no evidence for subsequent alkylacetylglycerol formation in the absence of NaF, but a small formation of radioactive PAF could be demonstrated from AAGP under the CPT assay conditions. The results of the CPT and acetyltransferase assays show a concentration of these activities particularly in rough microsomes and endoplasmic reticulum of nerve cell bodies and underline the potential for PAF synthesis de novo at these two sites.

Purification and properties of ribonuclelase E, an RNA-processing enzyme from Escherichia coli

The Escherichia coli RNA-processing enzyme RNAase E was purified through a number of steps including isoelectrofocusing. The final fraction contained mainly a single polypeptide of 66 kDa. However, while all the steps in the purification yielded the same qualitative activity, the specific activity of fractions as decreased in the last steps of the purification. By combining the most-purified enzyme with earlier fractions from the purification, we could show that the cells could contain a factor that enhances RNAase E activity. The purified enzyme showed the same characteristics with respect to temperature optimum, pH and ionic requirements as less-purified preparations. Testing specific inhibitors we concluded that the enzyme requires SH groups, free amino groups, and either of the amino acids tryptophan, tyrosine, histidine of methionine for its activity.

Incorporation of radioactive phospholipid into subclasses of high-density lipoproteins

The incorporation of orally administered phospholipid into plasma high-density lipoproteins (HDL) was studied in three subjects. Plasma was analyzed by equilibrium density gradient ultracentrifugation, 5, 6, and 8 h after ingestion of 1.1 g [3H-choline, 14C-dilinoleoyl]phosphatidylcholine. At all time points in all subjects, there was a peak of phosphatidylcholine specific activity in fractions of density approximately 1.10-1.13 g/ml, corresponding to the subclass previously designated HDL2a. There was also a more variable, smaller peak of specific activity of phospholipids in HDL2b (1.063-1.100 g/ml) and in fractions of density approximately 1.19 g/ml. In the 1.10-1.13 fraction, 97 and 71%, respectively, of the 3H and 14C radioactivity were in phospholipids. The 3H/14C ratio was similar in phospholipids of HDL subfractions, the d less than 1.07 fraction, and in the administered phospholipid. The results show preferential transfer or exchange or absorbed phosphatidylcholine into specific subclasses of HDL.

Platelet α Granules Contain a Growth Factor for Fibroblasts

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

Platelet alpha granules contain a growth factor for fibroblasts

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha- granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta- thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta- thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

Fractionation of humus-urease complexes

Some techniques commonly used for enzyme purification were unsuitable to purify urease extracted by pyrophosphate from soil. An all-or-none behaviour seemed to be a prominent feature of such experiments but in some instances slight increases of specific activity were observed. The most effective purification of soil urease was achieved by exhaustively ultrafiltrating the soil extract against 0.1 m pyrophosphate at pH 7.1, separating the retained material into fractions of mol. wt. higher (A I) and lower (A II) than 10 5, followed by gel chromatography. Increases in total activities were observed both after ultrafiltration and gel chromatography. Ultra-filtration increased the total activity of the extracted urease by about 8%. The specific activity of fraction A I increased four fold, that of fraction A II by more than three fold. Fractions obtained by gel chromatography accounted in toto for only 13.5% of the organic-C of the soil extract. Total urease activity increased by 45.6%. Specific activities increased to 6.9–18 times that of the soil extract.

Isolation and characterization of muscle membranes using surface-specific labels.

Membranes were purified from rat muscle by a differential centrifugation method that avoids the use of salt extraction or incubations at elevated temperature. Three populations of membrane-limited vesicles were defined having average densities of 1.112 (fraction I), 1.141 (fraction II), and 1.158 (fraction III) g/ml in a continuous sucrose gradient. Lactoperoxidase-catalyzed iodination of intact muscle prior to isolation of membranes resulted in highest specific activity in fraction I, although all fractions could be equally labeled after isolation. 125I-Labeled wheat germ agglutinin incubated at low concentration with intact muscle preferentially labeled fraction I. Parallel studies on previously isolated fractions indicated that fraction I also contained the highest concentration of potential receptors for wheat germ agglutinin. In experiments on whole muscle, concanavalin A bound predominantly to sarcolemma with slight variable binding to T-tubular and nuclear membrane but no binding to sarcoplasmic reticulum or mitochondria. Parallel binding studies with isolated membrane fragments indicated heavy binding of concanavalin A by membranes in fraction I with scattered binding in fractions II and III. Na+K+Mg2+ ATPase was specifically enriched in fraction I but was also present in fraction II in a proportion similar to 125I labeling. Ca2+ ATPase was most active in fraction II but present in significant levels in fraction I. It is concluded from these and other data that fraction I contains predominantly sarcolemma membrane, while T-tubular membrane may represent a significant component of fraction II. Ca2+ ATPase activity in fraction I is intrinsic to the sarcolemma.

Control of the increased protein synthesis in kidney of nephrotic rats by supernatant factors

1. The incorporation of [ 14C]phenylalanyl-tRNA into peptide was found to be the same in ribosomes from kidney of control and nephrotic rats that were incubated with excess poly(U) and a control liver transferase preparation. 2. The pH 5 supernatant fraction was prepared from kidney of control and nephrotic rats and incubated with control liver ribosomes. The incorporation of [ 14C]phenylalanyl-tRNA into peptide was two times higher with the nephrotic rat kidney preparation. 3. The increased incorporation could not be attributed to pH 7.8 alkaline ribonuclease activity nor its inhibitor but the increase was associated with aminoacyl-tRNA binding factor (transferase I) activity. 4. Kidney aminoacyl-tRNA binding factor (transferase I) was separated from peptidyl-tRNA translocation factor (transferase II) by Sephadex G-200. The incorporation of [ 14C]phenylalanyl-tRNA into peptide was measured using fractions containing transferase I obtained from control and nephrotic kidney and supplemented with control liver transferase II factor. Similarly fractions containing transferase II obtained from control and nephrotic kidney were supplemented with control liver transferase I factor. A significant increase was found in the specific activity of fractions containing transferase I from nephrotic kidney.

Isolation and purification of two phospholipases A from Bothrops venoms

Two proteins with phospholipase A activity (P-1 and P-2) were isolated from Bothrops neuwiedii venom by a procedure involving filtration through Sephadex G-50 at pH 4.5 (Step 1); filtration through Sephadex G-25 at pH 7.6 (Step 2); and chromatography on DEAE-Sephadex A-50 (or DEAE-cellulose) at pH 7.6 (Step 3). The developed method was adequate for 50–100 mg dried venom samples. When DEAE-Sephadex A-50 was used for Step 3, two symmetrical peaks of activity were eluted with constant specific activity throughout each peak (phospholipases A P-1 and P-2). P-1 and P-2 migrated as homogeneous monodisperse materials when subjected to polyacrylamide electrophoresis. When DEAE-cellulose was used for Step 3, three peaks of activity were eluted. The second and third peaks corresponded to P-1 and P-2 and represented 95% of the recovered enzyme activity; the first eluted fraction proved to be an artifact. Assay of P-1 or P-2 on pure ovolecithin as substrate yielded lysolecithin and fatty acid as sole reaction products, in accordance to the predicted stoichiometry. After Step 2, the phospholipase A activity recovered was higher than the total activity measured in the crude venom which is explained by the separation of an inhibitor during the enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with the crude venom. After Step 3, the minimal recovery of phospholipase A (P-1 plus P-2 activities) was about 80% and the purification achieved was 23-(P-1) and 19-fold (P-2) as compared with the crude venom activity. A purification method involving (1) an acidheat treatment, (2) a pH 9 fractionation, followed by (3) the Sephadex G-25 step, and (4) the DEAE-cellulose step gave also two different active proteins but both the total recovery of activity and the specific activity of fractions were less than those obtained with the standard method. Venoms of B. jararaca, B. jararacussu, and B. atrox were also fractionated and two phospholipases A fractions resembling P-1 and P-2 were obtained.

The glycosaminoglycans of dog compact bone and epiphyseal cartilage in the normal state and in experimental hyperparathyroidism.

In normal dogs and in dogs treated with 20 or 30 U.S.P. parathyroid extract for 5 and 3–4 days, respectively, the glycosaminoglycans of compact bone tissue were identified using the cetylpyridinium chloride precipitation method, and the concentrations of total hexosamines and the hexosamines corresponding to cetylpyridinium chloride precipitable acid glycosaminoglycans were determined. Further, the glycosaminoglycan pattern of the epiphyseal plate and the incorporation of35S-sulphate into the glycosaminoglycans of bone tissue and epiphyseal cartilage after administration of35S-sulphatein vivo was studied. In compact bone tissue, the hexosamines corresponding to acid glycosaminoglycans constuted approximately one third of the total hexosamine concentration and approximately 0.05–0.06% of the total dry weight. The main component of the acid glycosaminoglycans in bone was chondroitin-4-sulphate. This was sulphated to a higher degree and also of a higher molecular weight than thechondroitin sulphate of the epiphyseal cartilage, which in accordance with earlier investigations was found to have infrared characteristics of both chondroitin-4-sulphate and chondroitin-6-sulphate, with the former dominating. The molecular weights of the main part of bone chondroitin sulphate ranged from approximately 45,000 to 56,000. A small component of the bone glycosaminoglycans was hyaluronic acid. Large regularly recurring differences in the specific activity of fractions with differences in molecular weight in the condroitin sulphate of bone tissue and epiphyseal cartilage were noted. Treatment of the dogs with parathyroid extract gave no effect on the molecular weights of the chondroitin sulphate of the bone matrix or of the epiphyseal cartilage. Nor was there any unequivocal effect on the concentrations of total hexosamines or on the acid glycosaminoglycans. No evident stimulatory or depressant effect on the incorporation of35S-sulphate into the chondroitin sulphate or in the molecular distribution of newly sulphated and/or synthesized molecules of the condroitin sulphate within these tissues occurd.

Investigation of Homogeneous Isotope Exchange by Continuous Measurement of the Self-diffusion Process

HOMOGENEOUS isotope exchange is usually investigated by methods in which after a certain time both chemical forms of a given element are separated, specific activities of fractions are measured, and the dependence on time of the exchanged mole fraction is examined. There are many suitable methods of separation, but all have considerable drawbacks—for example, incomplete separation, need for long time intervals for separation during which supplementary exchange can occur, and zero-time exchanges as a result of exchange induced by co-precipitation or separation1.

Location of fractions of potassium in rabbit auricles

Sarcosomes and microsomes have been isolated from rabbit auricles by dispersion of the tissue in sucrose (0⋅28 M)-ethylene diaminetetra-acetate (0⋅01 M) and high-speed centrifugation at 0 to 1 °C. Of the total tissue potassium, about 10% was recovered from the 700 g -min fraction, which contained nuclei and coarse cell debris: 2 % from the 45000 or 250000 g -min fraction, which contained mainly sarcosomes and 0⋅7 % from the 750000 g -min fraction which contained mainly microsomes. When the auricles were immersed in physiological saline solution containing 42 K for 15 or 120 min before separation, the specific activity of the fractions differed, being highest in the particle-free suspending medium, intermediate in the sarcosomes and near to zero in the microsomes. The specific activity of the particle free fraction agreed closely and that of the sarcosomes moderately well with values predicted by analysis of the exchange in intact auricles. Dinitrophenol (10 -4 M) arrested uptake of 42 K by sarcosomes. Ouabain (10 -5 M) had no direct effect on the exchange. From measurements of electron micrographs it appeared that the sarcosomes occupied 8⋅2 ± 0⋅3 (S. E.) % of the intracellular volume. It is likely that the sarcosomes lost much of their potassium during extraction and probably contain most of the 21 % of the tissue potassium which exchanges slowly in the intact isolated tissue.